Little is known about the role of Notch signaling in esophageal squamous epithelial biology and diseases. In particular, there is a fundamental gap in understanding how Notch signaling contribute to normal esophageal epithelial squamous differentiation, carcinogenesis, and intratumoral cell heterogeneity, characterized by co- existing well-differentiated cell nests (i.e. keratin pearl) and disseminated, invasive poorly differentiated cells with mesenchymal traits found in esophageal squamous cell carcinoma (ESCC), one of the most deadly cancers. Continued existence of this gap represents an important problem because, until it is filled, mechanisms for Notch-mediated esophageal epithelial cell fate regulation will remain largely incomprehensible. The long-term goal is to better understand the molecular mechanisms by which tissue microenvironment influences malignant transformation of esophageal squamous epithelial cells. The objective in this proposal is to define the roles of Notch signaling in normal biology and tumor biology of the esophagus using innovative genetically engineered mouse models. The central hypothesis is that Notch induces squamous differentiation and senescence in a CSL-dependent manner. During tumor progression, microenvironmental cues activate a CSL-independent pathway to enrich migratory cancer stem cells (CSCs). This hypothesis has been formulated on the basis of preliminary data produced in the applicants' laboratories. The rationale for the proposed research is that, once it is known how Notch control cell fates, they can be manipulated pharmacologically, resulting in novel and innovative approaches in the prevention and treatment of ESCC. Guided by strong preliminary data, this hypothesis will be tested by pursuing three interrelated Specific Aims: (1) To elucidate how Notch activities contribute to the early stages of esophageal carcinogenesis in new mouse models; (2) To determine the roles of Notch for tumor progression in a conditional p120-catenin knockout mouse model; and (3) To delineate the roles of Notch in mouse CSCs in a novel orthotopic transplantation model where the tumor microenvironment is recapitulated. Genetic gain-of-function and loss-of-function experiments will be done in an inducible fashion in new mouse models. In vivo live imaging will be performed to assess the Notch-regulated CSC activities in vivo. The proposed research is significant, because it is expected to vertically advance and expand understanding of mechanisms by which esophageal epithelial homeostasis is regulated and in turn, deregulated in esophageal diseases, both benign and malignant, and potentially other squamous diseases in other tissue types, all through an appreciation of mouse pathobiology. Such knowledge will lead to development of innovative pharmacological strategies that will manipulate Notch signaling to alter esophageal cell fates and have the potential to advance in the therapy of ESCC and prevention of disease progression.